Jeffrey J. Clare

Production of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing multiple gene copies

We have constructed a synthetic secretion cassette encoding the alpha-factor prepro leader peptide from Saccharomyces cerevisiae fused to mouse epidermal growth factor (mEGF). This was used to compare the secretion of mEGF, a 53-amino acid polypeptide, in S. cerevisiae and Pichia pastoris. In both yeasts the leader sequence was accurately and efficiently cleaved showing that the S. cerevisiae-derived alpha-factor prepro region is correctly recognised and processed in P. pastoris. Of the total mEGF produced, over 90% was exported to the culture supernatant, although the final level of accumulation was dependent on the composition of the growth medium. With P. pastoris there was instability of the protein in minimal medium (yeast nitrogen base), probably caused by extracellular proteases. This was overcome by adding 1% Casamino acids and buffering the medium to pH 6.0. To increase the level of secreted mEGF we have developed a method for rapidly screening large numbers of P. pastoris transformants for the presence of many copies of a foreign gene. Using this procedure we isolated a strain containing 19 integrated copies of the mEGF gene which secreted 450 micrograms/ml of mEGF in high-density fermentations. Characterisation of the yeast-derived mEGF showed the presence of truncated forms, mEGF1-51 and mEGF1-52, as was found with S. cerevisiae-secreted human EGF [George-Nascimento et al., Biochemistry 27 (1988) 797-802]. In addition, the full-length protein, mEGF1-53, was secreted by P. pastoris.

Molecular Determinants of Voltage-dependent Gating and Binding of Pore-blocking Drugs in Transmembrane Segment IIIS6 of the Na+ Channel α Subunit

Mutations of amino acid residues in the inner two-thirds of the S6 segment in domain III of the rat brain type IIA Na+ channel (G1460A to I1473A) caused periodic positive and negative shifts in the voltage dependence of activation, consistent with an α-helix having one face on which mutations to alanine oppose activation. Mutations in the outer one-third of the IIIS6 segment all favored activation. Mutations in the inner half of IIIS6 had strong effects on the voltage dependence of inactivation from closed states without effect on open-state inactivation. Only three mutations had strong effects on block by local anesthetics and anticonvulsants. Mutations L1465A and I1469A decreased affinity of inactivated Na+ channels up to 8-fold for the anticonvulsant lamotrigine and its congeners 227c89, 4030w92, and 619c89 as well as for the local anesthetic etidocaine. N1466A decreased affinity of inactivated Na+ channels for the anticonvulsant 4030w92 and etidocaine by 3- and 8-fold, respectively, but had no effect on affinity of the other tested compounds. Leu-1465, Asn-1466, and Ile-1469 are located on one side of the IIIS6 helix, and mutation of each caused a positive shift in the voltage dependence of activation. Evidently, these amino acid residues face the lumen of the pore, contribute to formation of the high-affinity receptor site for pore-blocking drugs, and are involved in voltage-dependent activation and coupling to closed-state inactivation.

Voltage-gated sodium channels as therapeutic targets

Voltage-gated sodium channels (VGSCs) play a central role in the generation and propagation of action potentials in neurons and other cells. VGSC modulators have their origins in empirical pharmacology and are being used as local anaesthetics, antiarrhythmics, analgesics and antiepileptics, and for other disorders. However, the identification of a multigene family of VGSCs, along with tools to study the different subtypes in pathophysiology, is now providing a rational basis for selective intervention. Recent advances have addressed the technical challenges of expressing and assaying these complex proteins, enabling the correlation of empirical pharmacology to subtypes and the screening of individual subtypes for novel inhibitors with increased potency and selectivity.

Functional coupling of mammalian receptors to the yeast mating pathway using novel yeast/mammalian G protein alpha-subunit chimeras.

The expression of mammalian G protein coupled receptors (GPCRs) in S. cerevisiae provides a powerful assay system for functional analysis, ligand identification and pharmaceutical screening. However, relatively few receptors have been coupled to the pheromone response pathway via the yeast Gα, Gpa1p, or chimeric yeast/mammalian Gα subunits containing long C‐terminal regions of mammalian Gα proteins. We tested an extended range of seven such chimeras for Gα sub‐types of three major classes (Gαi/o, Gαs and Gαq), against eight human GPCRs (SST2, SST5, 5‐HT1A, 5‐HT1Dα, ML1B, P2Y1 and P2Y2). Although the Gαi/o chimeras increased the range of receptors that coupled efficiently, the Gαs and Gαq chimeras were inactive when expressed using the GPA1 promoter. We describe 10 novel Gpa1p chimeras, designated ‘transplants’, in which the C‐terminal five amino acids of Gpa1p were exchanged with mammalian residues. Coupling efficiency and ligand sensitivity improved significantly using the transplants. For the P2Y purinergic receptors, coupling could only be detected with the transplants; this is the first report of Gq specificity coupling in yeast. Thus, the transplants offer major advantages over previously described approaches, in terms of both the range of receptors coupled and the efficiency of coupling. Copyright

Comparative distribution of voltage-gated sodium channel proteins in human brain.

Antisera directed against unique peptide regions from each of the human brain voltage-gated sodium channel alpha subunits were generated. In immunoblots these were found to be highly specific for the corresponding recombinant polypeptides and to recognise the native holoprotein in human brain membrane preparations. These antisera were used to perform a comparative immunohistochemical distribution analysis of all four brain sodium channel subtypes in selected human CNS regions. Distinct but heterogeneous distribution patterns were observed for each of the alpha subunits. In general, these were complimentary to that previously shown for the corresponding human mRNAs. A high degree of conservation with respect to the distribution found in rat was also evident. The human alpha subunit proteins exhibited distinct subcellular localisation patterns. Types I, III and VI immunoreactivity was predominantly in neuronal cell bodies and proximal processes, whereas type II was concentrated along axons. This is similar to rat brain and suggests the different the sodium channel subtypes have distinct functions which are highly conserved between human and rodents. A notable difference was that the type III protein was detected in all human brain regions examined, unlike in rat brain where expression in adults is very restricted. Also in contrast to rat brain, the human type VI protein was not detected in axons of unmyelinated neurons. These differences may reflect true species variation and could have important implications for understanding the function of the sodium channel subtypes and their roles in human disease.

Distribution of voltage-gated sodium channel ?-subunit and ?-subunit mRNAs in human hippocampal formation, cortex, and cerebellum

The distribution of mRNAs encoding voltage‐gated sodium channel α subunits (I, II, III, and VI) and β subunits (β1 and β2) was studied in selected regions of the human brain by Northern blot and in situ hybridisation experiments. Northern blot analysis showed that all regions studied exhibited heterogenous expression of sodium channel transcripts. In situ hybridisation experiments confirmed these findings and revealed a predominantly neuronal distribution. In the parahippocampal gyrus , subtypes II and VI and the β‐subunit mRNAs exhibited robust expression in the granule cells of the dentate gyrus and pyramidal cell layer of the hippocampus. Subtypes I and III showed moderate expression in granule cells and low expression in the pyramidal cell layer. Distinct expression patterns were also observed in the cortical layers of the middle frontal gyrus and in the entorhinal cortex. In particular, all subtypes exhibited higher levels of expression in cortical layers III, V, and VI compared with layers I and II. All subtypes were expressed in the granular layer of the cerebellum , whereas specific expression of subtypes I, VI, β1, and β2 mRNAs was observed in Purkinje cells. Subtypes I, VI, and β1 mRNAs were expressed, at varying levels, in the pyramidal cells of the deep cerebellar nuclei. These data indicate that, as in rat, human brain sodium channel mRNAs have a distinct regional distribution, with individual cell types expressing different compliments of sodium channels. The differential distribution of sodium channel subtypes suggest that they have distinct roles that are likely to be of paramount importance in maintaining the functional heterogeneity of central nervous system neurons. J. Comp. Neurol. 422:123–139, 2000.

Cloning, distribution and functional analysis of the type III sodium channel from human brain.

The type III voltage‐gated sodium channel was cloned from human brain. The full‐length cDNA has 89% identity with rat type III, and the predicted protein (1951 amino acids) has 55 differences. The expression pattern of human type III mRNA was determined in adult brain tissue and, in contrast to rat, was detected in many regions, including caudate nucleus, cerebellum, hippocampus and frontal lobe. The human type III channel was stably expressed in Chinese hamster ovary (CHO) cells and its biophysical properties compared to the human type II channel using identical conditions. The voltage dependence and kinetics of activation were found to be similar to that of type II. The kinetics of inactivation of the two human subtypes were also similar. However, type III channels inactivated at more hyperpolarized potentials and were slower to recover from inactivation than type II. When expressed in human embryonic kidney (HEK293T) cells, type III channels produced currents with a prominent persistent component, which were similar to those reported for rat type II [Ma et al. (1997) Neuron, 19, 443–452]. However, unlike type II, this was prominent even in the absence of coexpressed G‐proteins, suggesting type III may adopt this gating mode more readily. The distinct properties of the channel, together with its wide distribution in adult brain, suggest that in humans, type III may have important physiological roles under normal, and perhaps also pathological conditions.

Recombinant Bordetella pertussis pertactin (P69) from the yeast Pichia pastoris: high-level production and immunological properties

Acellular whooping cough vaccines are based on pertussis toxoid but their effectiveness may be increased by the addition of other Bordetella pertussis antigens. We expressed the immunogenic outer membrane protein pertactin (P69) from B. pertussis to high levels in multi-copy transformants of the industrial yeast Pichia pastoris. In high-density fermentations, engineered P. pastoris yielded greater than 3 g of the protein per litre of culture. Purified recombinant pertactin was able to stimulate the incomplete protection afforded by toxoid to the level of the whole-cell vaccine, as shown by the Kendrick test, supporting its inclusion in future acellular vaccines.

Differential interactions of lamotrigine and related drugs with transmembrane segment IVS6 of voltage-gated sodium channels

Voltage-gated sodium channels are blocked by local anesthetic and anticonvulsant drugs. A receptor site for local anesthetics has been defined in transmembrane segment S6 in domain IV (IVS6) of the alpha subunit, but the anticonvulsant lamotrigine and related compounds have more complex structures than local anesthetics and may interact with additional amino acid residues. Apparent K(D) values for inactivated-state block of rat brain type IIA sodium channels expressed in Xenopus oocytes were 31.9 micro M, 17.3 micro M, 3.7 micro M and 10.3 micro M for lamotrigine and compounds 227c89, 4030w92 and 619c89, respectively. Compound 619c89 was the strongest frequency-dependent blocker, which correlated with higher affinity and a five-fold slower recovery from drug block compared to lamotrigine. Examination of lamotrigine block of mutant sodium channel alpha subunits, in which alanine had been substituted for each individual amino acid in IVS6, identified mutations I1760A, F1764A and Y1771A as causing the largest reductions in affinity (six-, seven- and 12-fold, respectively). The ratios of effects of these three mutations differed for compounds 227c89, 4030w92, and 619c89. The amino acid residues interacting with these pore-blocking drugs define a surface of IVS6 that is exposed to the pore and may rotate during gating.

Heterologous expression and functional analysis of rat NaV1.8 (SNS) voltage-gated sodium channels in the dorsal root ganglion neuroblastoma cell line ND7–23

The voltage-gated sodium channel NaV1.8 (SNS, PN3) is thought to be a molecular correlate of the dorsal root ganglion (DRG) tetrodotoxin resistant (TTX-R) Na+ current. TTX-R/NaV1.8 is an attractive therapeutic drug target for inflammatory and neuropathic pain on the basis of its specific distribution in sensory neurones and its modulation by inflammatory mediators. However, detailed analysis of recombinant NaV1.8 has been hampered by difficulties in stably expressing the functional protein in mammalian cells. Here, we show stable expression and functional analysis of rat NaV1.8 (rNaV1.8) in the rat DRG/mouse N18Tg2 neuroblastoma hybridoma cell line ND7-23. Rat NaV1.8 Na+ currents were recorded (789 +/- 89 pA, n=62, over 20-cell passages) that qualitatively resembled DRG TTX-R in terms of gating kinetics and voltage-dependence of activation and inactivation. The local anaesthetic drug tetracaine produced tonic inhibition of rNaV1.8 (mean IC50 value 12.5 microM) and in repeated gating paradigms (2-10 Hz) also showed frequency-dependent block. There was a correlation between the ability of several analogues of the anticonvulsant/analgesic compound lamotrigine to inhibit TTX-R and rNaV1.8 (r=0.72, P<0.001). RT-PCR analysis of wild type ND7-23 cells revealed endogenous expression of the beta1 and beta3 accessory Na+ channel subunits-the possibility that the presence of these subunits assists and stabilises expression of rNaV1.8 is discussed. We conclude that the neuroblastoma ND7-23 cell line is a suitable heterologous expression system for rNaV1.8 Na+ channels in that it allows stable expression of a channel with biophysical properties that closely resemble the native TTX-R currents in DRG neurones. This reagent will prove useful in the search for pharmacological inhibitors of rNaV1.8 as novel analgesics.